Solvent composition gradients are widely used in high performance liquid chromatography to circumvent the so called "general elution problem" encountered in isocratic (constant solvent composition) analysis where early eluting peaks are closely bunched and later peaks are spread progressively further apart in the chromatogram. By an appropriate choice of solvent gradient, it is often possible to retain the necessary resolution for separation of the early peaks while speeding the elution of the later peaks and, therefore, shortening the overall analysis time. The later peaks are also narrower under gradient elution, and diluted in less solvent so that detectability limits for these peaks are improved over isocratic conditions.
Versatile solvent gradient systems are available from several instrument manufacturing companies, but these systems tend to be considerably more expensive than isocratic solvent delivery systems. While these gradient solvent delivery systems offer great versatility, there are probably many applications where versatility could be sacrificed for cost as, for example, in dedicated analyzers, where the gradient profile could be optimized on one of the expensive gradient systems and transferred to a lower cost, less versatile system. For another perspective, gradient capability could be added to isocratic dedicated analyzers for little additional cost.
Gradient systems are usually classified as "low pressure" in which the gradient is formed on the inlet or low pressure side of the pump, or "high pressure" in which the gradient is formed on the outlet or high pressure side of two or more pumps. The low pressure gradient systems only require one high pressure pump, and are, therefore, usually less expensive than high pressure gradient systems. There are special problems that must be overcome in the design of a low pressure gradient system. The mechanically reciprocating, check valve type pumps commonly used in HPLC, draw solvent into the cylinder in discrete steps and tend to create a stair-step type gradient profile unless a detrimental mixing volume is placed downstream of the pump to smooth out these steps.
A more difficult problem can arise when switching valves are used to generate the gradient profile. If these valves do not operate at a high frequency compared to the pump frequency, or are not purposely synchronized with the pump frequency, random variation between the switching valve and the pump phases can generate significant departures from the intended gradient profile. The high pressure gradient systems place stringent requirements on the flow rate repeatability of the high pressure pumps near the end points of the gradient. Of particular importance is the flow rate repeatability at the lowest flow rate where mechanical, reciprocating check valve pumps tend to have problems because of check valve and main seal leakage. These problems have been solved to a satisfactory level for standard bore HPLC, where flow rates are apt to lie between 1/2 and 5 milliliters per minute, but their solution has added to the cost of the gradient solvent delivery systems.
While mechanically reciprocating check valve pumps dominate in standard bore HPLC, the low flow rates of microbore HPLC (typically between 1 and 100 microliters per minute) strain the present state of check valve technology, and, as a result, single stroke syringe pumps that do not require check valves are most common. Low pressure gradient formation is not practical with these pumps because of their large hold-up volume. The high gradient formation by mixing flow from two or more syringe pumps can be a problem with some commonly used gradients such as methanol/water and acetonitrile/water because of a peculiar oscillating mode that develops from differences in viscosity and compressibility of the two solvents and their mixtures. This oscillating mode can be eliminated by the use of a constant back pressure valve downstream of the point of solvent mixing and ahead of the column, or the use of individual back pressure regulating valves at the output of each pump.
While not used in commercial HPLC gradient solvent delivery systems of current vintage, various mixing chamber type gradient generators have been reported in the literature. These devices use a stirred mixing chamber of fixed or variable volume that is initially filled with the first solvent of the gradient. The second end point solvent of the gradient is pumped into this mixing chamber to change its composition with time, and, thus, generate the solvent gradient. The simplest of these is the exponential dilution chamber which has a fixed volume. Unfortunately, the exponential dilution chamber generates a convex upward gradient profile that is generally undesirable in HPLC. This type of profile is steepest at the beginning of this separation where the peaks are already more tightly bunched in time, and levels out in the last part of the separation where it is usually desirable to bring widely spaced peaks closer together.
More desirable profiles are obtained theoretically by connecting a number of exponential dilution chambers in series. Equations for the gradient profiles of one, two, three, and four exponential dilution chambers connected in series are commonly known to those skilled in the art. These equations have been used to calculate the curves plotted in Graph I. As the numbers of chambers increases, the initial steepness of the curves become less and the curves approach the shape of the error function plotted in Graph II. It will be clearly understood by those skilled in the art that the error function is the shape of the concentration profile in frontal chromatographic analyses where the sample is introduced at the head of the column as a steep function.
It would be desirable to generate the desirable error function type gradient, approximated with a large number of exponential dilution chamber in series, by a packed bed. A packed bed would be much less expensive than a series of exponential dilution chambers and much more reliable.
A packed-bed gradient could consist of some sort of column filled with suitable packing material such as millimeter size chemically inert glass beads. The relatively large beads would offer little flow resistance and the pressure drop across the bed would be low for the flow rates used in HPLC. The millimeter sized beads would be of the order of 1000 times larger than the micrometer sized particles used in efficient HPLC columns. Pressure drop varies proportionally to the inverse square of the particle diameter. Thus, the pressure drop across a packed-bed gradient generating column would be of the order of one million times less than that across a chromatographic column if they are of comparable length and diameter.
Because of its inherent low pressure drop the packed-bed gradient generating column could be placed on the low pressure, inlet side of the pump, as well as on the high pressure, outlet side of the pump. Low pressure operation of the packed bed would allow use of less expensive low pressure fittings and valves, and use of a low pressure column. A transparent glass column might be used for visibility of the bed. On the other hand, high pressure operation would provide a built in pulse dampener, due to compressibility of the solvent in the bed, to dampen the pulses from lower cost pulsating pumps. The pulse dampening feature could be enhanced by the use of a flexible wall column such as a bourdon tube. The high pressure gradient capability of the packed bed generator makes it compatible with high pressure syringe pumps used in microbore HPLC.